In general, the invention features methods for modifying nucleic acid substrates, for example, for the production of RNA-protein fusions.
Covalently bonded RNA-protein fusions may be used in methods for generating or isolating proteins with desired properties from pools of proteins. To create such fusions, an RNA and the peptide or protein that it encodes may be joined during in vitro translation using synthetic RNA that carries a peptidyl acceptor, such as puromycin, at its 3'-end (Roberts & Szostak (1997) Proc. Natl. Acad. Sci. USA 94, 12297-12302). In this process, the synthetic RNA, which is devoid of stop codons, is typically synthesized by in vitro transcription from a DNA template followed by 3'-ligation to a DNA linker carrying puromycin. The DNA sequence causes the ribosome to pause at the end of the open reading frame, providing additional time for the puromycin to accept the nascent peptide chain and resulting in the production of the RNA-protein fusion molecule.